Maintenance of genetic stability relies not only on faithful repair of endogenous and exogenous DNA damage, but also on the prompt activation of checkpoints leading to cell cycle arrest and apoptosis, when damage exceeds a critical threshold. Spontaneous deamination of cytosine and 5-methylcytosine to uracil and thymine at CpG sites is a major mechanism of endogenous mutagenesis and plays an important role in tumorigenesis. Several uracil and thymine DNA N-glycosylases active on G: U and G: T mismatches are present in mammalian cells and protect from deamination events at CpG sites. We identified one of these enzymes, human MED1 (also known as MBD4), as an interact or of the mismatch repair (MMR) protein MLH1. Inactivation MED1 in the mouse germ line increases mutagenicity at CpG sites, but only 3-fold, raising the possibility of compensation by back-up repair activities. One such candidate is the enzyme TDG that has in vitro biochemical activities similar to MED1. Furthermore, the general MMR system may, at least in principle, act on G: T and G: U mismatches. At the moment, the relative contributions of MED1, TDG and MMR to the repair of G: U and G: T mismatches at CpG sites in vivo are unclear. Recently, we identified a novel role of MED1 in the DNA damage response to alkylating agents and other anti-tumor drugs. MED1-/- MEFs treated with increasing doses of alkylating agents, like MNNG, and other anti-tumor drugs, failed to undergo cell cycle arrest and apoptosis. Much like MMR-defective cells, resistance of MED1-/- MEFs to MNNG was due to a tolerance mechanism, as DNA damage accumulated but did not elicit a G2-M checkpoint and p53 activation. MMR proteins levels are markedly reduced in MED1-/- MEFs, suggesting that MED1 may be required for alkylating agent cytotoxicity by maintaining integrity of the MMR signaling complex. We hypothesize that MED1 has a dual role in DNA repair and DNA damage response. Experiments in this proposal are designed to: 1) characterize the role of MED1 in G2-M cell cycle arrest and apoptosis induced by DNA damage; 2) examine the role of MED1 in maintaining integrity of MMR protein levels; 3) examine the roles of MED1, TDG, and MMR in the repair of G: T mismatches at CpG sites in vivo. These studies will provide new insights into mechanisms of endogenous mutagenesis, response to DNA damage and resistance of cancer cells to anti-tumor chemotherapy. [unreadable] [unreadable]